GLUTATHIONE TRANSFERASES-DEFINING STRUCTURE & FUNCTION

描述（由申请方提供）：谷胱甘肽转移酶催化谷胱甘肽（GSH）硫与各种携带亲电官能团的内源性和异源性底物的亲核加成。它们是哺乳动物中烷化剂代谢和解毒中最重要的酶。此外，它们代表了一个具有多种未开发功能的蛋白质超家族。本计画的目标是了解谷胱甘肽转移酶及其同源物在原核生物和真核生物生物中的作用。这一目标将通过阐明分子结构和功能之间的关系来实现。调查将集中在一些哺乳动物和细菌酶的结构和催化机制方面知之甚少。将实现四个具体目标。首先，谷胱甘肽转移酶的结构，动力学和催化作用将通过以下方式阐明：（i）使用酰胺质子交换质谱法来探测μ类酶中控制配体释放速率的特定结构元件的动力学（ii）将酰胺-质子交换质谱法扩展到毫秒时间域;和（iii）通过串联质谱法将质子交换动力学映射到单个残基。其次，选择GSI-I转移酶同源物在大肠杆菌蛋白质组将进行调查，以确定其独特的生物学作用。本论文将通过以下几个方面对谷胱甘肽转移酶超家族EGST、b2989和yibF的功能进行研究：（i）基因产物的克隆、表达和机理分析;（ii）同源物在大肠杆菌中的表达谱分析;大肠杆菌蛋白质组在不同的环境条件下，通过二维差异凝胶电泳和质谱;（iii）删除选定的基因，以确定表型反应;和（iv）基因产物的结构测定。第三，将通过以下方式研究κ类和相关的GSI-I转移酶：（i）测定大鼠κ类酶的三维结构，（ii）分析酶的底物特异性，和（iii）恶臭假单胞菌萘分解代谢途径中密切相关的2-羟基色烯-2-羧酸异构酶的机理分析。最后，机制和结构的微粒体酶的调查将延长;（一）独特的机械行为的活化酶的分析;（二）的结构和动力学的探索的本地和活化形式的酶在洗涤剂溶液中的酰胺质子交换质谱法;和（iii）酰胺质子交换分析的二维晶体质谱法。

DESCRIPTION (provided by applicant): The glutathione transferases catalyze the nucleophilic addition of the sulfur of glutathione (GSH) to a wide variety of endogenous and xenobiotic substrates bearing electrophilic functional groups. They are the single most important enzyme in the metabolism and detoxication of alkylating agents in mammals. In addition, they represent a superfamily of proteins with diverse and unexplored functions. It is the goal of this project to understand the role of glutathione transferases and their homologues in the biology of both prokaryotes and eukaryotes. This goal will be accomplished through the elucidation of the relationship between molecular structure and function. The investigations will focus on poorly understood aspects of the structure and catalytic mechanism of selected mammalian and bacterial enzymes. Four specific aims will be pursued. First, the structure, dynamics and catalysis of glutathione transferases will be elucidated through; (i) the use amide-proton exchange mass spectrometry to probe the dynamics of specific structural elements in the class mu enzymes that control the rate of release of ligands (products) from the active site; (ii) extension of amide-proton exchange mass spectrometry into the millisecond time domain; and (iii) mapping of proton exchange kinetics to individual residues by tandem mass spectrometry. Second, selected GSI-I transferase homologues in the Escherichia coil proteome will be investigated to determine their unique biological roles. The functions of three members of the GSH transferase superfamily EGST, b2989 and yibF will be explored by; (i) cloning, expression and mechanistic characterization of the gene products; (ii) profiling the expression of the homologues in the E. coli proteome under different environmental conditions by two-dimensional difference gel electrophoresis and mass spectrometry; (iii) deletion of selected genes to determine phenotypic responses; and (iv) structure determination of the gene products. Third, class kappa and related GSI-I transferases will be investigated through (i) determination of the three dimensional structure of the class kappa enzyme from rat, (ii) analysis of the substrate specificity of the enzyme, and (iii) mechanistic analysis of the closely related 2-hydroxychromene-2-carboxylate isomerase in the naphthalene catabolic pathway of Pseudomonas putida. Finally, mechanistic and structural investigations of the microsomal enzyme will be extended by; (i) analysis of the unique mechanistic behavior of the activated enzyme; (ii) an exploration of the structure and dynamics of the native and activated forms of the enzyme in detergent solution by amide-proton exchange mass spectrometry; and (iii) amide-proton exchange analysis of two-dimensional crystals by mass spectrometry.